The University of Southern Mississippi The Aquila Digital Community Dissertations Spring 5-2012 Genetic Variation in Potentially Virulent Vibrio parahaemolyticus From the Northern Gulf of Mexico Nicholas Felix Noriea III University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/dissertations Part of the Aquaculture and Fisheries Commons, Biology Commons, Immunology and Infectious Disease Commons, Marine Biology Commons, and the Terrestrial and Aquatic Ecology Commons Recommended Citation Noriea, Nicholas Felix III, "Genetic Variation in Potentially Virulent Vibrio parahaemolyticus From the Northern Gulf of Mexico" (2012). Dissertations. 799. https://aquila.usm.edu/dissertations/799 This Dissertation is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Dissertations by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. The University of Southern Mississippi GENETIC VARIATION IN POTENTIALLY VIRULENT VIBRIO PARAHAEMOLYTICUS FROM THE NORTHERN GULF OF MEXICO by Nicholas Felix Noriea III Abstract of a Dissertation Submitted to the Graduate School of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2012 ABSTRACT GENETIC VARIATION IN POTENTIALLY VIRULENT VIBRIO PARAHAEMOLYTICUS FROM THE NORTHERN GULF OF MEXICO By Nicholas Felix Noriea III May 2012 Vibrio parahaemolyticus (Vp) is a gram-negative bacterium found naturally in marine and estuarine environments. Vp is found in oysters including those which are later consumed by the public. Sub-populations of potentially virulent Vp contain specific virulence factors and are relevant human pathogens capable of causing gastroenteritis, wound infection, and death. The tdh and trh genes, both encoding hemolysins, have been correlated with the majority of clinical Vp isolates but have not been shown to be the definitive virulence factors. A total of 146 Vp isolates from the northern Gulf of Mexico were collected and probed for the presence of specific virulence factors such as tdh, trh, Type III secretion System (T3SS) 1, T3SS2α, T3SS2β, and toxRS. The most representative sub-group of potentially pathogenic Vp were tdh+/trh+. Isolates containing only tdh were uncommon, and tdh-/trh+ isolates were extremely rare. The distribution of virulence factors in environmental Vp strains was compared with strain fitness through Vp human intestinal epithelial cell cytotoxicity assays and oyster (Crassostrea virginica) hemocyte bactericidal challenges. The assays were performed to determine significant differences in cytotoxicity or bactericidal survival between pathogen subgroups (tdh+/trh-, tdh-/trh+, and tdh-/trh+). ii Significant changes in Vp gene expression were measured for representative clinical strains of the diametric virulence factor sub-groups tdh+/trh- and tdh-/trh+. Transcriptomic changes were measured during exposure to human intestinal epithelial cells and oyster hemocyte. Relative changes in the Vp transcriptome were compared both over time and between strain to give significantly regulated genes involved in cytotoxicity and oyster hemocyte bactericidal avoidance. iii COPYRIGHT BY NICHOLAS FELIX NORIEA III 2012 The University of Southern Mississippi GENETIC VARIATION IN POTENTIALLY VIRULENT VIBRIO PARAHAEMOLYTICUS FROM THE NORTHERN GULF OF MEXICO by Nicholas Felix Noriea III A Dissertation Submitted to the Graduate School of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Approved: _Darrell J. Grimes_____________________ Director _Crystal N. Johnson____________________ _Robert J. Griffitt______________________ _Mohamed O. Elasri____________________ _Kevin S. Dillon_______________________ _Susan A. Siltanen_____________________ Dean of the Graduate School May 2012 ACKNOWLEDGMENTS The author would like to thank the committee chair, Jay Grimes for providing all of the many opportunities and support to explore and conduct this project. The author would also like to thank each of the committee members, Dr. Kevin Dillon, Dr. Mohamed Elasri, Dr. Joe Griffitt, and Dr. Crystal Johnson (LSU) for their support, guidance, and assistance throughout the project. Particularly, the author would like to thank Dr. Joe Griffitt for assistance in microarray analysis and Dr. Crystal Johnson for training and access to a large collection of Vp isolates. Special thanks go to Drs. Lou and Karen Burnett (NOAA) for hemocyte extraction training, Dr. Wes Burkhardt (Medical Gentics) for use of cell culturing equipment, Dr. Walt Conley (SUNY-Potsdam) for guidance, and Dr.Andy DePaola (FDA) for guidance and clinical isolates. Finally, the author would like to thank Idrissa Boube, Kim Griffitt, and Dawn Rebarchik for assistance and support throughout the project. iv TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii ACKNOWLEDGMENTS .................................................................................................. iv LIST OF TABLES ............................................................................................................ vii LIST OF ILLUSTRATIONS ........................................................................................... viii CHAPTER I. INTRODUCTION ..................................................................................... 1 II. DISTRIBUTION OF VIRULENCE FACTORS IN V. PARAHAEMOLYTICUS FROM THE NORTHERN GULF OF MEXICO .................................................................................. 9 Materials and Methods Results Discussion About This Chapter III. VARIATION IN CYTOTOXICITY AND SURIVIVAL RATES OF POTENTIALLY VIRULENT V. PARAHAEMOLYTICUS ..................... 25 Materials and Methods Results Discussion IV. TRANSCRIPTIONAL REGULATION IN POTENTIALLY VIRULENT V. PARAHAEMOLYTICUS DURING EXPOSURE TO INTESTINAL EPITHELIAL CELLS AND OYSTER HEMOLYMPH ........................................................................................ 37 Materials and Methods Results Discussion V. CONCLUSIONS AND FUTURE DIRECTIONS ................................... 65 APPENDIXES .................................................................................................................. 67 REFERENCES .................................................................................................................. 96 v LIST OF TABLES Table 1. Source of clinical Vp isolates used in this study .................................................. 11 2. Distribution of virulence factors in environmental isolates (n=146) ................... 15 3. Distribution of virulence factors in clinical isolates (n=12) ................................. 15 4. Vp survival in Oyster Hemocyte .......................................................................... 29 5. Percentage of Caco-2 cell death after exposure to Vp .......................................... 30 6. Student t-test p-values for Vp virulence factor sub-group comparison in oyster hemocyte bactericidal assays ................................................................................ 32 7. Student t-test p-values for Vp virulence factor sub-group comparisons in Caco-2 cytotoxicity assays ................................................................................................ 32 8. Summary of all genes significantly up or down-regulated in each Vp challenge group ..................................................................................................................... 46 9. Total genes significantly up or down-regulated by category. .............................. 52 10. Total summary of genes displaying significant expression variation between strains at equal time points/challenge groups .................................................................. 52 vi LIST OF ILLUSTRATIONS Figure 1. Locations of the four sampling sites along the Mississippi Gulf Coast ................ 10 2. Representative images of Vp isolates using RING-FISH for T3SS2α gene vopB2 ............................................................................................................................... 17 3. The percent distribution of potentially pathogenic Vp .......................................... 18 4. The percentage of Vp cells still viable after four-hour oyster hemocyte bactericidal assays, separated by potential pathogen subgroup. ............................................... 30 5. The percentage of Caco-2 cells killed after a four-hour Vp-Caco-2 cytotoxicity assay ...................................................................................................................... 31 6. Vp growth over time (minutes) when exposed to Caco-2 cells in EMEM with 10% FBS ........................................................................................................................ 41 7. Vp growth over time (minutes) in oyster hemolymph assays ............................... 41 8. Total genes regulated in Vp-oyster hemolymph challenges .................................. 47 9. Total genes regulated in Vp-Caco-2 challenges .................................................... 48 10. Ratio of significantly up-regulated to down-regulated genes in oyster hemolymph challenges for each functional gene category and challenge type......................... 49 11. Ratio of significantly up-regulated